1. Field of the Invention
The present invention relates to an apparatus for and method of measuring bio-chips, and more particularly to an apparatus for and method of measuring bio-chips, which can implement an illumination method of a novel type that illuminates a bio sample (which may be also referred to as a “bio specimen”) through a side face of a substrate using a diffusion plate so as to more efficiently measure fluorescence information of a bio-chip over a wide field of view.
2. Background of the Related Art
In general, a bio-chip refers to a chip including the arrangement of biological molecules such as DNA molecule, etc. The bio-chip is typically configured such that very small cells of a micro unit are plotted in the form of a matrix on a predetermined substrate.
A glass plate is generally used as the substrate and a substrate made of other material such as silicone may be used. A variety of DNA molecules are distributed on cells of an ultra-fine size.
A bio-chip measurement apparatus is an instrument that is manufactured specially to measure fluorescence emitted from fine cells of a micro unit formed on the bio-chip and image the emitted fluorescence as an image. Such a bio-chip measurement apparatus enables acquisition of fluorescence observation image from micro cells and readout of information on the cells.
The bio-chip measurement apparatus can be classified into two types in terms of a measurement principle. One is a scanning-type measurement apparatus, and the other is such a measurement apparatus that observes an area of a to-be-measured object at a time such as a camera. In case of the former, information is sequentially read out from cells to cell.
In case of the latter, all the cells on the bio-chip covered by a field of view of a microscope are concurrently analyzed.
This type of bio-chip measurement apparatus is a merit that fluorescence information is obtained on a parallel basis from all the cells as to-be-measured objects, it is possible analyze data a relatively high speed as compared to the scanning type bio-chip measurement apparatus.
Moreover, since the latter type of bio-chip measurement apparatus generally has a relatively simple structure and is simple to operate as compared to the scanning-type bio-chip measurement apparatus, it is suitable for analysis of a large amount of data.
The latter type of bio-chip measurement apparatus adopting a total internal reflection illumination method is illustrated as its representative example in FIG. 1.
As shown in FIG. 1, a parallel-collimated excitation light emitted from a light source 10 is incident to a side face 13 of a substrate 12 on which bio samples 14 are placed. In this case, the bio-chip 11 is configured such that bio samples 14 are arranged on a thin transparent substrate 12 having a rectangular parallelepiped shape and a front face 15 on which bio samples 14 are arranged, a rear face 16 and a side face 13.
At this time, the excitation light emitted from the light source substrate is incident to the side face of the substrate at an incidence angle which can be defined through total internal reflection between the front face on which the bio samples are arranged and the rear face opposite to the front face. Meanwhile, fluorescence is observed from the bio samples by means of an evanescent field formed over the front face of the substrate on which the bio samples are arranged. The fluorescence observation image is observed and measured by a fluorescence detecting section 18, so that fluorescence information can be measured from the bio-chip.
However, such a conventional bio-chip measurement apparatus has a limitation in illumination to the bio sample when it is desired to measure fluorescence information of the bio-chip over a wide field of view.
If it is required that fluorescence information of the bio-chip should be measured over a wide field of view, a main factor that must be taken into consideration in the bio-chip measurement apparatus is uniformity of light with which to illuminate the bio samples to receive an identical signal from the same points positioned in various portions within a field of view for observation.
A main reason why brightness of light is ununiform is that flux of light illuminated is not even.
For example, as shown in FIG. 2, from the brightness distribution in a cross-section of a green laser beam excited and oscillated by a laser diode used in a bio-chip measurement apparatus using Cy3 as fluorescent dye, it can be seen that the brightness of the green laser beam is not uniform over the cross-section of the laser beam.
The brightness of the light is maximum at the central portion of the beam and is gradually decreased as it goes toward the peripheral portion of the beam.
In FIG. 2, the photo at the left side shows the brightness of a laser beam excited and oscillated by a laser diode and the graph at the right side shows brightness intensity of the laser beam.
Here, a white colored circle in the photo indicates a region where the brightness of the laser beam is as low as twice the maximum value.
In addition, in case where a laser beam is, at a predetermined inclination angle, incident to the front surface of the substrate on which samples of the bio-chip to be measured are placed in order to use the total internal reflection illumination method such as the above conventional bio-chip measurement apparatus, regions which are illuminated with the light are indicated with them divided into black-colored areas which progress at a certain incidence angle, which leads to a difficulty in illuminating the bio samples when it is desired to measure fluorescence information of the bio-chip over a wide field of view.